Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois 60612, USA.
Mol Cell Proteomics. 2010 Sep;9(9):1804-18. doi: 10.1074/mcp.M110.000075. Epub 2010 May 5.
The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.
肌球蛋白马达的分子构象受重链、轻链、肌球蛋白结合蛋白 C 和肌联蛋白之间的分子间相互作用调节，并受翻译后修饰（PTMs）控制。胶内消化后结合 LC/MS/MS 经典地用于鉴定心肌肌节 PTM；然而，这种方法受到蛋白质大小、等电点和肽提取困难的限制。我们报告了一种基于溶液的工作流程，用于全面分离内源性心肌肌节蛋白，重点是调节轻链（RLC），其中磷酸化的特定位点尚不清楚。亚细胞分级分离后采用 OFFGEL 电泳导致肌节蛋白的内源性电荷变体（包括调节和必需轻链、肌球蛋白重链和粗丝上的肌球蛋白结合蛋白 C）得以分离。使用反相 HPLC 分离和 UV 检测进一步纯化 RLC，在完整蛋白质水平上富集 RLC PTM，并提供内源性 RLC 电荷变体的化学计量和定量评估。消化后进行 LC/MS/MS 分析，明确鉴定出聚焦在独特 OFFGEL 电泳馏分中的内源性 RLC 电荷变体未磷酸化（78.8%）、单磷酸化（18.1%）和双磷酸化（3.1%）RLC。本研究的新颖之处在于：1）聚焦毫克量的内源性心肌肌节亚蛋白组，分辨率可与二维电泳相媲美，2）在完整蛋白质水平上实现了翻译后修饰变体的分离和定量，3）在溶液中实现了完整的高分子量粗丝蛋白的分离，4）分离了完整的 RLC 电荷变体；在小鼠中鉴定到一种新型的双磷酸化形式，在人类非衰竭和衰竭心脏样本中鉴定到并定量了单磷酸化、单脱酰胺和脱酰胺/磷酸化形式，从而证明了该方法的临床实用性。
